Figure 3. Effects of sPD-1 and sTim-3 on the frequency of IFN-γ spot-forming cells and the percentage of responses to each antigen in mice immunized with rAd5-SIV vaccine. (A) The frequency of IFN-γ spot-forming cells specific for SIV structural proteins Gag, Pol, and Env. (B) The frequency of IFN-γ spot-forming cells specific for SIV non-structural proteins Nef, Vpx, Vpr, Vif, Rev, and Tat, respectively (C) The frequency of IFN-γ spot-forming cells specific for all SIV antigens. (D) The percentage of IFN-γ spot-forming cells specific for structural antigens Gag, Pol, Env, and nonstructural antigens (N3VRT: Nef, Vpx, Vpr, Vif, Rev, and Tat) were shown in a pie chart. C57BL/6 mice were immunized with either rAd5-SIV alone or rAd5-SIV co-administered with rAd5-sPD1 or rAd5-sTim3, or rAd5-SIV co-administered with both rAd5-sPD1 and rAd5-sTim3. At 2 wk after immunization, splenocytes were harvested and cultured with SIV peptides for each antigen and subjected to an IFN-γ ELISPOT assay. The data were analyzed by two-way ANOVA. The bars represent the standard errors. *P < 0.05; **P < 0.01; ***P < 0.001. The data are the representation of two independent experiments.